Comparative sequence analysis of the complete set of 40S ribosomal proteins in the Senegalese sole (Solea senegalensis Kaup) and Atlantic halibut (Hippoglossus hippoglossus L.) (Teleostei: Pleuronectiformes): phylogeny and tissue- and development-specific expression

Background  

Ribosomal proteins (RPs) are key components of ribosomes, the cellular organelle responsible for protein biosynthesis in cells. Their levels can vary as a function of organism growth and development; however, some RPs have been associated with other cellular processes or extraribosomal functions. Their high representation in cDNA libraries has resulted in the increase of RP sequences available from different organisms and their proposal as appropriate molecular markers for phylogenetic analysis.     

Regulation of nucleolar signalling to p53 through NEDDylation of L11

Several studies have shown that ribosomal proteins (RPs) are important mediators of p53 activation in response to nucleolar disruption; however, the pathways that control this signalling function of RPs are currently unknown. We have recently shown that RPs are targets for the ubiquitin‐like molecule NEDD8, and that NEDDylation protects RPs from destabilization. Here, we identify NEDD8 as a crucial regulator of L11 RP signalling to p53. A decrease in L11 NEDDylation during nucleolar stress causes relocalization of L11 from the nucleolus to the nucleoplasm. This not only provides the signal for p53 activation, but also makes L11 susceptible to degradation. Mouse double minute 2 (MDM2) ‐mediated NEDDylation protects L11 from degradation and this is required for p53 stabilization during nucleolar stress. By controlling the correct localization and stability of L11, NEDD8 acts as a crucial, new regulator of nucleolar signalling to p53.     

Deregulation of ribosomal proteins in human cancers

The ribosome, the site for protein synthesis, is composed of ribosomal RNAs (rRNAs) and ribosomal proteins (RPs). The latter have been shown to have many ribosomal and extraribosomal functions. RPs are implicated in a variety of pathological processes, especially tumorigenesis and cell transformation. In this review, we will focus on the recent advances that shed light on the effects of RPs deregulation in different types of cancer and their roles in regulating the tumor cell fate.     

蛋白酶体调节颗粒的结构生物学特征及其功能

蛋白酶体调节颗粒(regulatory particle,RP)参与调控许多重要信号通路的蛋白质降解,在维持细胞稳态中发挥重要作用.近年来,真核细胞蛋白酶体在癌症治疗中的作用机制及药物研发已引起了广泛关注,并有3种蛋白酶体抑制剂已用于临床治疗.随着蛋白酶体功能研究的不断深入,以及晶体学和冷冻电镜技术在其结构生物学研究中...     

Toward an Integrated Structural Model of the 26S Proteasome

The 26S proteasome is the end point of the ubiquitin-proteasome pathway and degrades ubiquitylated substrates. It is composed of the 20S core particle (CP), where degradation occurs, and the 19S regulatory particle (RP), which ensures substrate specificity of degradation. Whereas the CP is resolved to atomic resolution, the architecture of the RP is largely unknown. We provide a comprehensive analysis of the current structural knowledge on the RP, including structures of the RP subunits, physical protein-protein interactions, and cryoelectron microscopy data. These data allowed us to compute an atomic model for the CP-AAA-ATPase subcomplex. In addition to this atomic model, further subunits can be mapped approximately, which lets us hypothesize on the substrate path during its degradation.The ubiquitin-proteasome pathway is the major route used by eukaryotic cells for the disposal of misfolded or damaged proteins and for controlling the lifespan of proteins (1–3). As a consequence, the ubiquitin-proteasome pathway regulates a plethora of fundamental cellular processes, such as protein quality control, DNA repair, and signal transduction (4). The 26S proteasome is a molecular machine of ∼2.5 MDa that targets polyubiquitylated proteins. It comprises two subcomplexes, the 20S core particle (CP)¹ and one or two copies of asymmetric 19S regulatory particles (RPs), which bind to the end(s) of the barrel-shaped CP.The active sites of the proteasome are located in the CP cavity where proteolytic cleavage of substrates takes place. Electron microscopy (EM) and x-ray crystallography have revealed that the CP is a cylinder consisting of four concentrically stacked rings (5–7): two identical “α”-rings, each assembled of seven homologous proteins, form the outer rings, and two identical “β”-rings, also assembled of seven homologs, form the two inner rings. Proteolysis is confined to the cavity formed by the β-rings, a nanocompartment sequestered from the cytosol.The RPs regulate substrate degradation by (i) binding polyubiquitylated substrates, (ii) subsequently deubiquitylating them, (iii) substrate unfolding, and (iv) opening the “gate” to the CP (8). The RPs consists of six AAA-ATPase subunits and at least 13 non-ATPase subunits. In contrast to the CP, the architecture of the RP subunits remains largely unknown. The problems that hamper structural characterization of the RP are manifold. It has proven difficult to obtain homogeneous, concentrated preparations of 26S proteasomes or RPs because the RP tends to disassociate into heterogeneous subcomplexes during purification and concentration. Moreover, many of the RP subunits likely exhibit a significant degree of structural variability. As a consequence, x-ray crystallographic analysis of the entire RP has not been accomplished to date, and only a few subunit fragments have been amenable to high resolution structure determination.For cryo-EM and protein-protein interaction experiments, the requirements for sample homogeneity are less stringent. Recently, the Drosophila melanogaster 26S proteasome was resolved to ∼20 Å (9). Various proteomics approaches have led to proposals for topological maps of the RP (10–13). The resolution of protein-protein interaction data typically corresponds to the diameters of the proteins or domains found to interact, which are typically far beyond 20 Å. Because of the limited resolution, neither cryo-EM maps nor protein-protein interaction networks are by themselves sufficient to determine the RP architecture (i.e. the localization of the RP subunits in the complex).The integration of atomic models, cryo-EM maps, and protein-protein interaction data is currently the most promising approach to resolve the architecture of the 26S proteasome (14–18). Here, we provide a comprehensive analysis of the current structural knowledge on the RP, including structures of RP subunits, physical protein-protein interactions, and cryo-EM data. Based on these data, we provide a model for the CP-AAA-ATPase subcomplex. Finally, we outline a path toward resolving the architecture of the 26S proteasome by an integrative structure determination approach, which in turn will provide a basis for a mechanistic understanding.     

Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana

Agroinfiltrated Nicotiana benthamiana is a flexible and scalable platform for recombinant protein (RP) production, but its great potential is hampered by plant proteases that degrade RPs. Here, we tested 29 candidate protease inhibitors (PIs) in agroinfiltrated N. benthamiana leaves for enhancing accumulation of three unrelated RPs: glycoenzyme α‐Galactosidase; glycohormone erythropoietin (EPO); and IgG antibody VRC01. Of the previously described PIs enhancing RP accumulation, we found only cystatin SlCYS8 to be effective. We identified three additional new, unrelated PIs that enhance RP accumulation: N. benthamiana NbPR4, NbPot1 and human HsTIMP, which have been reported to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of all three RPs is enhanced by each PI similarly, suggesting that the mechanism of degradation of unrelated RPs follows a common pathway. Inhibitory functions HsTIMP and SlCYS8 are required to enhance RP accumulation, suggesting that their target proteases may degrade RPs. Different PIs additively enhance RP accumulation, but the effect of each PI is dose‐dependent. Activity‐based protein profiling (ABPP) revealed that the activities of papain‐like Cys proteases (PLCPs), Ser hydrolases (SHs) or vacuolar processing enzymes (VPEs) in leaves are unaffected upon expression of the new PIs, whereas SlCYS8 expression specifically suppresses PLCP activity only. Quantitative proteomics indicates that the three new PIs affect agroinfiltrated tissues similarly and that they all increase immune responses. NbPR4, NbPot1 and HsTIMP can be used to study plant proteases and improve RP accumulation in molecular farming.     

Insights into the Mechanism of Ribosomal Incorporation of Mammalian L13a Protein during Ribosome Biogenesis

In contrast to prokaryotes, the precise mechanism of incorporation of ribosomal proteins into ribosomes in eukaryotes is not well understood. For the majority of eukaryotic ribosomal proteins, residues critical for rRNA binding, a key step in the hierarchical assembly of ribosomes, have not been well defined. In this study, we used the mammalian ribosomal protein L13a as a model to investigate the mechanism(s) underlying eukaryotic ribosomal protein incorporation into ribosomes. This work identified the arginine residue at position 68 of L13a as being essential for L13a binding to rRNA and incorporation into ribosomes. We also demonstrated that incorporation of L13a takes place during maturation of the 90S preribosome in the nucleolus, but that translocation of L13a into the nucleolus is not sufficient for its incorporation into ribosomes. Incorporation of L13a into the 90S preribosome was required for rRNA methylation within the 90S complex. However, mutations abolishing ribosomal incorporation of L13a did not affect its ability to be phosphorylated or its extraribosomal function in GAIT element-mediated translational silencing. These results provide new insights into the mechanism of ribosomal incorporation of L13a and will be useful in guiding future studies aimed at fully deciphering mammalian ribosome biogenesis.     

Ribosomal proteins of Thermus thermophilus fused to beta-galactosidase are imported into the nucleus of eukaryotic cells

Archaea, Bacteria, and Eukarya have 34 homologous ribosomal protein (RP) families in common. Comparisons of published amino acid sequences prompted us to question whether RPs of the prokaryote Thermus thermophilus contain nuclear localization signals (NLSs), which are recognized by the nuclear import machinery of eukaryotic cells and are thereby translocated into the nucleoplasm ultimately accumulating in the nucleolus. Several RPs of T. thermophilus - specifically S12, S17, and L2 - were selected for this study since their three-dimensional structures as well as rRNA interaction patterns are precisely known at the molecular level. Fusion proteins of these RPs were constructed and subsequently expressed in COS cells. N-terminally tagged fusions with dimeric EGFP and C-terminally tagged hybrids with beta-galactosidase of prokaryotic RP S17 (S17p) were targeted to the nucleoplasm where they were visualized by direct fluorescence and by indirect immune staining, respectively. A region containing the classical monopartite NLS KRKR, which is known to physically interact with karyopherin alpha2, was delineated by tagging specific S17p fragments with beta-galactosidase. Unexpectedly, S12p and L2p hybrids accumulated in the nucleolus. Due to their size, RPs tagged with beta-galactosidase can only be imported into the nucleus when NLS-recognition is mediated by karyopherins since they are otherwise excluded from entry into the nucleoplasm of eukaryotic cells. Our results indicate that after the formation of the nuclear compartment during evolution, the newly established eukaryotic cell relied on the pre-existing basic amino acid clusters of the prokaryotic RPs for use as NLSs.     

Ribosomal Proteins RPL37, RPS15 and RPS20 Regulate the Mdm2-p53-MdmX Network

Changes to the nucleolus, the site of ribosome production, have long been linked to cancer, and mutations in several ribosomal proteins (RPs) have been associated with an increased risk for cancer in human diseases. Relevantly, a number of RPs have been shown to bind to MDM2 and inhibit MDM2 E3 ligase activity, leading to p53 stabilization and cell cycle arrest, thus revealing a RP-Mdm2-p53 signaling pathway that is critical for ribosome biogenesis surveillance. Here, we have identified RPL37, RPS15, and RPS20 as RPs that can also bind Mdm2 and activate p53. We found that each of the aforementioned RPs, when ectopically expressed, can stabilize both co-expressed Flag-tagged Mdm2 and HA-tagged p53 in p53-null cells as well as endogenous p53 in a p53-containing cell line. For each RP, the mechanism of Mdm2 and p53 stabilization appears to be through inhibiting the E3 ubiquitin ligase activity of Mdm2. Interestingly, although they are each capable of inducing cell death and cell cycle arrest, these RPs differ in the p53 target genes that are regulated upon their respective introduction into cells. Furthermore, each RP can downregulate MdmX levels but in distinct ways. Thus, RPL37, RPS15 and RPS20 regulate the Mdm2-p53-MdmX network but employ different mechanisms to do so.     

The plastid ribosomal proteins. Identification of all the proteins in the 50 S subunit of an organelle ribosome (chloroplast)

We have completed identification of all the ribosomal proteins (RPs) in spinach plastid (chloroplast) ribosomal 50 S subunit via a proteomic approach using two-dimensional electrophoresis, electroblotting/protein sequencing, high performance liquid chromatography purification, polymerase chain reaction-based screening of cDNA library/nucleotide sequencing, and mass spectrometry (reversed-phase HPLC coupled to electrospray ionization mass spectrometry and electrospray ionization mass spectrometry). Spinach plastid 50 S subunit comprises 33 proteins, of which 31 are orthologues of Escherichia coli RPs and two are plastid-specific RPs (PSRP-5 and PSRP-6) having no homologues in other types of ribosomes. Orthologues of E. coli L25 and L30 are absent in spinach plastid ribosome. 25 of the plastid 50 S RPs are encoded in the nuclear genome and synthesized on cytosolic ribosomes, whereas eight of the plastid RPs are encoded in the plastid organelle genome and synthesized on plastid ribosomes. Sites for transit peptide cleavages in the cytosolic RP precursors and formyl Met processing in the plastid-synthesized RPs were established. Post-translational modifications were observed in several mature plastid RPs, including multiple forms of L10, L18, L31, and PSRP-5 and N-terminal/internal modifications in L2, L11 and L16. Comparison of the RPs in gradient-purified 70 S ribosome with those in the 30 and 50 S subunits revealed an additional protein, in approximately stoichiometric amount, specific to the 70 S ribosome. It was identified to be plastid ribosome recycling factor. Combining with our recent study of the proteins in plastid 30 S subunit (Yamaguchi, K., von Knoblauch, K., and Subramanian, A. R. (2000) J. Biol. Chem. 275, 28455-28465), we show that spinach plastid ribosome comprises 59 proteins (33 in 50 S subunit and 25 in 30 S subunit and ribosome recycling factor in 70 S), of which 53 are E. coli orthologues and 6 are plastid-specific proteins (PSRP-1 to PSRP-6). We propose the hypothesis that PSRPs were evolved to perform functions unique to plastid translation and its regulation, including protein targeting/translocation to thylakoid membrane via plastid 50 S subunit.     

Peptides that activate the 20S proteasome by gate opening increased oxidized protein removal and reduced protein aggregation

The proteasome is a multicatalytic protease that is responsible for the degradation of the majority of intracellular proteins. Its role is correlated with several major regulatory pathways that are involved in cell cycle control, signaling, and antigen presentation, as well as in the removal of oxidatively damaged proteins. Although several proteasomal catalytic inhibitors have been described, very few activators have been reported to date. Some reports in the literature highlight the cellular protective effects of proteasome activation against oxidative stress and its effect on increased life span. In this work, we describe a peptide named proteasome-activating peptide 1 (PAP1), which increases the chymotrypsin-like proteasomal catalytic activity and, consequently, proteolytic rates both in vitro and in culture. PAP1 proteasomal activation is mediated by the opening of the proteasomal catalytic chamber. We also demonstrate that the observed proteasomal activation protected cells from oxidative stress; further, PAP1 prevented protein aggregation in a cellular model of amyotrophic lateral sclerosis. The role of 20SPT gate opening underlying protection against oxidative stress was also explored in yeast cells. The present data indicate the importance of proteasomal activators as potential drugs for the treatment of pathologies associated with the impaired removal of damaged proteins, which is observed in many neurodegenerative diseases.     

Modifications of glyceraldehyde-3-phosphate dehydrogenase induced by increasing concentrations of peroxynitrite: early recognition by 20S proteasome

Peroxynitrite, a potent oxidizing and nitrating species, induces covalent modifications of biomolecules in a number of pathological conditions. In previous studies with S. cerevisiae, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as being especially susceptible to nitration by peroxynitrite. The activity of this enzyme was strongly inhibited by low doses of peroxynitrite in yeast and in cultured rat astrocytes. Here, the sequence of modifications of isolated mammalian GAPDH induced by increasing concentrations of peroxynitrite is demonstrated to be as follows: (i) oxidation, leading to inactivation and to enhanced susceptibility of GAPDH for proteasomal degradation, (ii) oligomer formation, and (iii) nitration. In our study the susceptibility for degradation by isolated 20S proteasome was by far the most sensitive parameter for peroxynitrite-induced damage to GAPDH, implying that this might also occur under pathological conditions where peroxynitrite is generated at low concentrations in vivo.     

Systematic chromosomal deletion of bacterial ribosomal protein genes

Detailed studies of ribosomal proteins (RPs), essential components of the protein biosynthetic machinery, have been hampered by the lack of readily accessible chromosomal deletions of the corresponding genes. Here, we report the systematic genomic deletion of 41 individual RP genes in Escherichia coli, which are not included in the Keio collection. Chromosomal copies of these genes were replaced by an antibiotic resistance gene in the presence of an inducible, easy-to-exchange plasmid-born allele. Using this knockout collection, we found nine RPs (L15, L21, L24, L27, L29, L30, L34, S9, and S17) nonessential for survival under induction conditions at various temperatures. Taken together with previous results, this analysis revealed that 22 of the 54 E. coli RP genes can be individually deleted from the genome. These strains also allow expression of truncated protein variants to probe the importance of RNA-protein interactions in functional sites of the ribosome. This set of strains should enhance in vivo studies of ribosome assembly/function and may ultimately allow systematic substitution of RPs with RNA.     

Specificity and Regulation of the Endoplasmic Reticulum‐Associated Degradation Machinery

The endoplasmic reticulum‐associated degradation (ERAD) machinery selects native and misfolded polypeptides for dislocation across the ER membrane and proteasomal degradation. Regulated degradation of native proteins is an important aspect of cell physiology. For example, it contributes to the control of lipid biosynthesis, calcium homeostasis and ERAD capacity by setting the turnover rate of crucial regulators of these pathways. In contrast, degradation of native proteins has pathologic relevance when caused by viral or bacterial infections, or when it occurs as a consequence of dysregulated ERAD activity. The efficient disposal of misfolded proteins prevents toxic depositions and persistent sequestration of molecular chaperones that could induce cellular stress and perturb maintenance of cellular proteostasis. In the first section of this review, we survey the available literature on mechanisms of selection of native and non‐native proteins for degradation from the ER and on how pathogens hijack them. In the second section, we highlight the mechanisms of ERAD activity adaptation to changes in the ER environment with a particular emphasis on the post‐translational regulatory mechanisms collectively defined as ERAD tuning.